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| 6. |
- Lerche, Michael, et al.
(författare)
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Structure and Cooperativity of the Cytosolic Domain of the CorA Mg2+ Channel from Escherichia coli
- 2017
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 25:8, s. 1175-1186.e4
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Tidskriftsartikel (refereegranskat)abstract
- Structures of the Mg2+ bound (closed) and apo (open) states of CorA suggests that channel gating is accomplished by rigid-body motions between symmetric and asymmetric assemblies of the cytosolic portions of the five subunits in response to ligand (Mg2+) binding/unbinding at interfacial sites. Here, we structurally and biochemically characterize the isolated cytosolic domain from Escherichia coli CorA. The data reveal an Mg2+-ligand binding site located in a novel position between each of the five subunits and two Mg2+ ions trapped inside the pore. Soaking experiments show that cobalt hexammine outcompetes Mg2+ at the pore site closest to the membrane. This represents the first structural information of how an analog of hexa-hydrated Mg2+ (and competitive inhibitor of CorA) associates to the CorA pore. Biochemical data on the isolated cytoplasmic domain and full-length protein suggests that gating of the CorA channel is governed cooperatively.
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| 7. |
- Lloris-Garcerá, Pilar, et al.
(författare)
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Why Have Small Multidrug Resistance Proteins Not Evolved into Fused, Internally Duplicated Structures?
- 2014
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 426:11, s. 2246-2254
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Tidskriftsartikel (refereegranskat)abstract
- The increasing number of solved membrane protein structures has led to the recognition of a common feature in a large fraction of the small-molecule transporters: inverted repeat structures, formed by two fused homologous membrane domains with opposite orientation in the membrane. An evolutionary pathway in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers has been posited. A gene duplication event enables the evolution of two oppositely orientated proteins that form antiparallel heterodimers. Finally, fusion of the two genes generates an internally duplicated transporter with two oppositely orientated membrane domains. Strikingly, however, in the small multidrug resistance (SMR) family of transporters, no fused, internally duplicated proteins have been found to date. Here, we have analyzed fused versions of the dual-topology transporter EmrE, a member of the SMR family, by blue-native PAGE and in vivo activity measurements. We find that fused constructs give rise to both intramolecular inverted repeat structures and competing intermolecular dimers of varying activity. The formation of several intramolecularly and intermolecularly paired species indicates that a gene fusion event may lower the overall amount of active protein, possibly explaining the apparent absence of fused SMR proteins in nature.
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| 9. |
- Ndi, Mama, et al.
(författare)
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Structural basis for Cbp3 interaction with newly synthesized cytochrome b during mitochondrial respiratory chain assembly
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:45, s. 16663-16671
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Tidskriftsartikel (refereegranskat)abstract
- Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3–Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol–cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.
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| 10. |
- Palombo, Isolde, 1984-, et al.
(författare)
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The Periplasmic Loop Provides Stability to the Open State of the CorA Magnesium Channel
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:33, s. 27547-27555
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Tidskriftsartikel (refereegranskat)abstract
- Crystal structures of the CorA Mg2+ channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open metal free state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg2+. We find that when Mg2+ is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg2+ is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.
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| 11. |
- Palombo, Isolde, et al.
(författare)
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Why Is the GMN Motif Conserved in the CorA/Mrs2/Alr1 Superfamily of Magnesium Transport Proteins?
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:28, s. 4842-4847
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Tidskriftsartikel (refereegranskat)abstract
- Members of the CorA/Mrs2/Alr1 superfamily of transport proteins mediate magnesium uptake in all kingdoms of life. Family members have a low degree of sequence conservation but are characterized by a conserved extracellular loop. While the degree of sequence conservation in the loop deviates to some extent between family members, the GMN family signature motif is always present. Structural and functional data imply that the loop plays a central role in magnesium selectivity, and recent biochemical data suggest it is crucial for stabilizing the pentamer in the magnesium-free (putative open) conformation. In this study, we present a detailed structure-function analysis of the extracellular loop of CorA from Thermotoga maritima, which provides molecular insight into how the loop mediates these two functions. The data show that loop residues outside of the GMN motif can be substituted if they support the pentameric state, but the residues of the GMN motif are intolerant to substitution. We conclude that G(312) is absolutely required for magnesium uptake, M-313 is absolutely required for pentamer integrity in the putative open conformation, and N-314 plays a role in both of these functions. These observations suggest a molecular reason why the GMN motif is conserved throughout the CorA/Mrs2/Alr1 superfamily.
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| 13. |
- Rapp, Mikaela, et al.
(författare)
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Emulating membrane protein evolution by rational design
- 2007
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 315:5816, s. 1282-1284
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Tidskriftsartikel (refereegranskat)abstract
- How do integral membrane proteins evolve in size and complexity? Using the small multidrug-resistance protein EmrE from Escherichia coli as a model, we experimentally demonstrated that the evolution of membrane proteins composed of two homologous but oppositely oriented domains can occur in a small number of steps: An original dual-topology protein evolves, through a gene-duplication event, to a heterodimer formed by two oppositely oriented monomers. This simple evolutionary pathway can explain the frequent occurrence of membrane proteins with an internal pseudo–two-fold symmetry axis in the plane of the membrane.
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| 14. |
- Rapp, Mikaela, et al.
(författare)
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Experimentally based topology models for E. coli inner membrane proteins
- 2004
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 13:4, s. 937-945
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Tidskriftsartikel (refereegranskat)abstract
- Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.
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| 15. |
- Rapp, Mikaela, et al.
(författare)
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Identification and evolution of dual-topology membrane proteins
- 2006
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 13:2, s. 112-116
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Tidskriftsartikel (refereegranskat)abstract
- Integral membrane proteins are generally believed to have unique membrane topologies. However, it has been suggested that dual-topology proteins that adopt a mixture of two opposite orientations in the membrane may exist. Here we show that the membrane orientations of five dual-topology candidates identified in Escherichia coli are highly sensitive to changes in the distribution of positively charged residues, that genes in families containing dual-topology candidates occur in genomes either as pairs or as singletons and that gene pairs encode two oppositely oriented proteins whereas singletons encode dual-topology candidates. Our results provide strong support for the existence of dual-topology proteins and shed new light on the evolution of membrane-protein topology and structure.
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| 16. |
- Rapp, Mikaela, 1974-
(författare)
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The Ins and Outs of Membrane Proteins : Topology Studies of Bacterial Membrane Proteins
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- α-helical membrane proteins comprise about a quarter of all proteins in a cell and carry out a wide variety of essential cellular functions. This thesis is focused on topology analyses of bacterial membrane proteins. The topology describes the two-dimensional structural arrangement of a protein relative to the membrane. By combining large-scale experimental and bioinformatics techniques we have produced experimentally constrained topology models for the major part of the Escherichia coli membrane proteome. This represents a substantial increase in available topology information for bacterial membrane proteins. Many membrane protein structures show signs of internal duplication and approximate two-fold in-plane symmetry. We propose a step-wise pathway to explain how proteins with such internal inverted repeats have evolved. The pathway is based on the ‘positive-inside’ rule and starts with a protein that can adopt two topologies in the membrane, i.e. a “dual” topology protein. The gene encoding the dual topology protein is duplicated and eventually, through re-distribution of positively charge residues, the two resulting homologous proteins become fixed in opposite orientations in the membrane. Finally, the two proteins may fuse into one single polypeptide with an internal inverted repeat structure. Finally, we re-create the proposed step-wise evolutionary pathway in the laboratory by showing that only a small number of mutations are required in order to transform the homo-dimeric, dual topology protein EmrE into a hetero-dimeric complex composed of two oppositely oriented proteins.
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| 17. |
- Seppälä, Susanna, et al.
(författare)
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Control of Membrane Protein Topology by a Single C-Terminal Residue
- 2010
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 328:5986, s. 1698-1700
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism by which multispanning helix-bundle membrane proteins are inserted into their target membrane remains unclear. In both prokaryotic and eukaryotic cells, membrane proteins are inserted cotranslationally into the lipid bilayer. Positively charged residues flanking the transmembrane helices are important topological determinants, but it is not known whether they act strictly locally, affecting only the nearest transmembrane helices, or can act globally, affecting the topology of the entire protein. Here we found that the topology of an Escherichia coli inner membrane protein with four or five transmembrane helices could be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C terminus. This observation points to an unanticipated plasticity in membrane protein insertion mechanisms.
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| 18. |
- Tidemand Johansen, Nicolai, et al.
(författare)
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Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation
- 2022
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Ingår i: eLIFE. - 2050-084X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.
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